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J. R. Soc. Interface (2009) 6, 435–446
doi:10.1098/rsif.2008.0348
Published online 30 September 2008
Structure and properties of
strontium-doped phosphate-based glasses
Ensanya A. Abou Neel1, Wojciech Chrzanowski1, David M. Pickup2,
Luke A. O’Dell3, Nicola J. Mordan1, Robert J. Newport2,
Mark E. Smith3 and Jonathan C. Knowles1,*
1
Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute,
256 Gray’s Inn Road, London WC1X 8LD, UK
2
School of Physical Sciences, University of Kent, Canterbury CT2 7NH, UK
3
Department of Physics, University of Warwick, Coventry CV4 7AL, UK
Owing to similarity in both ionic size and polarity, strontium (Sr2C) is known to behave in a
comparable way to calcium (Ca2C), and its role in bone metabolism has been well
documented as both anti-resorptive and bone forming. In this study, novel quaternary
strontium-doped phosphate-based glasses, containing 1, 3 and 5 mol% SrO, were synthesized
and characterized. 31P magic angle spinning (MAS) nuclear magnetic resonance results
showed that, as the Sr2C content is increased in the glasses, there is a slight increase in
disproportionation of Q 2 phosphorus environments into Q1 and Q3 environments. Moreover,
shortening and strengthening of the phosphorus to bridging oxygen distance occurred as
obtained from FTIR. The general broadening of the spectral features with Sr2C content is
most probably due to the increased variation of the phosphate–cation bonding interactions
caused by the introduction of the third cation. This increased disorder may be the cause of the
increased degradation of the Sr-containing glasses relative to the Sr-free glass. As confirmed
from elemental analysis, all Sr-containing glasses showed higher Na2O than expected and this
also could be accounted for by the higher degradation of these glasses compared with Sr-free
glasses. Measurements of surface free energy (SFE) showed that incorporation of strontium
had no effect on SFE, and samples had relatively higher fractional polarity, which is not
expected to promote high cell activity. From viability studies, however, the incorporation of
Sr2C showed better cellular response than Sr2C-free glasses, but still lower than the positive
control. This unfavourable cellular response could be due to the high degradation nature of
these glasses and not due to the presence of Sr2C.
Keywords: phosphate-based glasses; biocompatibility; surface free energy; ion release;
FTIR spectroscopy; nuclear magnetic resonance
1. INTRODUCTION
water in a few hours to those that are stable for years
(Drake 1985; Drake & Allen 1985).
PBGs containing Ca2C and NaC ions have
potential applications in both soft- and hard-tissue
engineering, since the ions released from these glasses
are natural constituents of the human body and are
therefore tolerated by it (Knowles 2003). It has been
shown that extracts from the less soluble glass
compositions (i.e. glasses with high calcium oxide
content) enhanced cell proliferation and upregulated
gene expression (Franks et al. 2002; Bitar et al. 2004).
Moreover, the cells were able to attach, spread and
proliferate in a manner comparable to the positive
control along with the formation of a collagen-rich
mineralized matrix (Gough et al. 2002). It has been
reported that calcium ions play a key role in cell
activation mechanisms, thereby controlling many
growth-associated processes and functional activities
of cells (Salih et al. 2000).
Phosphate-based glasses (PBGs) are inorganic polymers composed of PO3K
tetrahedra that form the
4
backbone of the structure and metal cations that
charge balance the phosphate chains (Knowles et al.
2001; Skelton et al. 2007). Glasses can be prepared
containing various metal oxides such as CuO (Abou
Neel et al. 2005a), Fe2O3 (Yu et al. 1997; Ahmed et al.
2004; Abou Neel et al. 2005b), Al2O3 (Shah et al. 2005),
TiO2 (Rajendran et al. 2006; Abou Neel et al. 2007;
Abou Neel & Knowles 2008) or ZnO (Salih et al. 2007;
Abou Neel et al. 2008) to suit the end applications.
Furthermore, Drake and Allen found that PBGs with
a suitable composition dissolve in water with a zeroorder rate, and, by tailoring the composition further,
it was possible to produce glasses with stabilities
ranging from those that would completely degrade in
*Author for correspondence ([email protected]).
Received 7 August 2008
Accepted 5 September 2008
435
This journal is q 2008 The Royal Society
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436 Strontium-doped phosphate-based glasses
E. A. Abou Neel et al.
Table 1. Glass compositions (including abbreviation used in the text hereafter), processing temperatures, density and glass
transition temperature.
glass composition (mol%)
properties
glass code
notation
calcium oxide sodium oxide
phosphorus
pentoxide
strontium
oxide
r (g cmK3)
Tg (8C)
P50C30N20
P50C30N19Sr1
P50C30N17Sr3
P50C30N15Sr5
CNP
CNPSr1
CNPSr3
CNPSr5
30
30
30
30
50
50
50
50
0
1
3
5
2.58G0.00
2.59G0.01
2.60G0.00
2.62G0.02
383G0.7
391G6
421G4
428G3
20
19
17
15
Physiologically, strontium and calcium are remarkably similar; they are both absorbed in the gastrointestinal tract, concentrated in bone and excreted primarily
in urine (Wren et al. 2008). One of the mechanisms of
strontium incorporation into bone involves ionic
exchange with bone calcium. Owing to these similarities,
strontium is currently used in bone therapies (Llinas
et al. 2006). For example, strontium ranelate (SR) is
marketed under the name of Protelos, Osseor, Protos,
Bivalos, Protaxos and Ossum, and used to treat
osteoporosis. It has been shown that SR strengthens
bone, increases bone mass and density and lessens the
possibility of vertebral and hip fracture in elderly
women. This has been attributed to the fact that the
human body absorbs strontium as if it were calcium.
Since strontium has higher atomic weight than calcium,
this accounts for the increasing bone density (Meunier
et al. 2004; Reginster et al. 2005). Moreover, the
radioactive isotopes of strontium (89Sr and 85Sr) are
used to treat scattered painful bone metastases that
affect two-thirds of patients with advanced and metastatic cancers (Giammarile et al. 1999; Saarto et al. 2002).
In this study, calcium phosphate glasses containing
strontium oxide for potential use in bone tissue
regeneration have been prepared. Starting with a base
composition of (CaO)0.3(Na2O)0.2(P2O5)0.5, the Na2O
was replaced with 1, 3 and 5 mol% SrO. The objective
of this study was to evaluate the effect of strontium
oxide additions on the structure (X-ray powder
diffraction, 23Na and 31P magic angle spinning (MAS)
nuclear magnetic resonance (NMR) and FTIR spectroscopies), bulk (density, thermal analysis, degradation
and ion release), and surface properties (wettability,
surface free energy (SFE) and biocompatibility) of
these glasses.
2. EXPERIMENTAL
2.1. Glass preparation
Glass rods of 15 mm diameter were prepared as per the
compositions given in table 1. Sodium dihydrogen
orthophosphate ( NaH2PO 4), calcium carbonate
(CaCO3), phosphorus pentoxide (P2O5) and strontium
carbonate (SrCO3; BDH, Poole, UK) were used as
precursors (all chemicals were above 98% purity).
These precursors were weighed out and placed into a
Pt/10% Rh crucible (Type 71040, Johnson Matthey,
Royston, UK). The crucible and contents were heated
in a furnace (Carbolite, RHF 1500, Sheffield, UK) at
11008C for 1 hour. The resultant melt was then poured
J. R. Soc. Interface (2009)
into a preheated split graphite mould that was in turn
placed in an annealing furnace at 3508C for 1 hour to
remove any residual stresses in the glass. The mould
was then allowed to cool slowly to room temperature
in the annealing furnace overnight. The rods were
cut into discs of approximately 1 mm thickness using
a Testbourne diamond saw with methanol as a
coolant/lubricant.
Powdered samples for thermal and structural
characterization were prepared by grinding pieces of
glass using a zirconia mill.
2.2. Structural characterization
2.2.1. X-ray powder diffraction. The powdered samples
were annealed in a ceramic pot from room temperature
to the crystallization temperature, obtained from the
differential thermal analyser, at a heating rate of
208C minK1 using Lenton Furnace (Lenton Thermal
Designs LTD, UK). The temperature was maintained
at that temperature for 3 hours to ensure proper
crystallization, and finally it was reduced to room
temperture at the same rate used for heating. X-ray
diffraction (XRD) data for these crystallized samples
were collected on a Bruker D8 Advance diffractometer
(Brüker, UK) in flat-plate geometry, using Ni-filtered
Cu Ka radiation and a Bruker Lynx Eye detector. The
X-ray patterns were measured from 10 to 1008 2q with a
step size of 0.028 and a count time of 0.1 s. The phases
were identified using CRYSTALLOGRAPHICA SEARCHMATCH software (Oxford Cryosystems, Oxford, UK)
and the International Centre for Diffraction Data
(ICDD) database (vols 1–42).
2.2.2. Nuclear magnetic resonance. 23Na MAS NMR
experiments were conducted at 7.05 T (23Na Larmor
frequency 97.4 MHz) with a Chemagnetics Infinity Plus
spectrometer, using a 4 mm diameter rotor spinning at
12.5 kHz. Aqueous NaCl was used as a reference, with
the sharp resonance from this set to 0 ppm. The liquid
908 pulse length was determined to be 2.5 ms, although a
much shorter pulse length (0.5 ms) was used on the solid
samples. A one-pulse sequence was used, with a preacquisition delay of 7.5 ms and a recycle delay of 5 s,
which was sufficient to produce fully relaxed spectra.
The overall experiment time was approximately 15 min
per sample. Certain spectra were also recorded at
14.1 T on a Bruker Avance II spectrometer with similar
experimental conditions to those above.
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Strontium-doped phosphate-based glasses
31
P MAS NMR experiments were also conducted at
7.05 T (31P Larmor frequency 121.5 MHz) using a 4 mm
diameter rotor spinning at 12.5 kHz. Solid NH4H2PO4
was used as a secondary reference compound and the
signal from this set to 0.9 ppm. A pulse length of 1.5 ms
was used (corresponding to an approx. 308 tip angle),
with a pre-acquisition delay of 7.5 ms and a recycle
delay of 5 s. Overall experiment time was approximately 10 min per sample.
All NMR spectra were subsequently processed using
Spinsight and fitted using either dmfit2007 (Massiot
et al. 2002) or QuadFit (Kemp 2004).
2.2.3. FTIR spectroscopy. FTIR spectra were recorded
in transmission mode on a Biorad FTS175C spectrometer controlled by WIN-IR software. Samples were
diluted in dry KBr and scanned in the range of 4000–
450 cmK1. Each spectrum was the result of summing
64 scans.
2.2.4. Elemental analysis. The crystalline powdered
samples were carbon coated before being examined
under a JEOL 5410LV scanning electron microscope
( JEOL UK Ltd, UK) equipped with an INCA 300,
EDX System (Oxford, UK) energy dispersive X-ray
spectroscopy accessory. The electron beam was produced with a 10 kV accelerating voltage and directed
at the coated specimens. X-rays, produced by the
elements in the specimen, were directed to the liquid
nitrogen-cooled detector that allowed the identification of elements on the basis of their characteristic
X-ray energies.
2.3. Bulk glass characterization
2.3.1. Density measurements. Density measurements
were conducted in triplicate on each sample by the
Archimedes method, using an analytical balance
(Mettler Toledo, UK) with an attached density kit.
Owing to the soluble nature of the glass compositions
investigated, ethanol was used as the immersion liquid
for these measurements.
2.3.2. Thermal characterization. The glass transition
temperature (Tg) was determined using a Pyris Diamond DSC (DIAMDSC, Perkin-Elmer Instruments,
Shelton, USA). Moreover, crystallization temperature
(Tc) and melting temperature (Tm) were also carried out
using a Setaram Differential Thermal Analyser (TG
DTA/DSC Labsys, Setaram, Caluire, France) as
described previously (Abou Neel et al. 2008).
2.3.3. Degradation studies. The surface area of the glass
discs was calculated from the dimensions measured
with Mitutoyo Digimatic Vernier Callipers. Glass discs
were placed in glass bottles containing 25 ml of highpurity water with pH adjusted with few drops of
NH4OH to 7G0.2. At various time points (0, 1, 4, 7, 11
and 15 days), the solution was removed for ion release
analysis. Then the discs were taken out of their
respective containers, and excess moisture was removed
J. R. Soc. Interface (2009)
E. A. Abou Neel et al. 437
by blotting the discs dry with tissue paper before
weighing. The discs were then placed in a fresh solution
of deionized water and placed back into the incubator at
378C. These studies were conducted in triplicate, and the
data plotted as cumulative degradation, i.e. percentage
weight loss per unit area, as a function of time for the
various glass compositions.
2.3.4. Ion release measurements. Ion release studies
were simultaneously conducted, and the medium was
analysed for cation (Ca2C, NaC and Sr2C) and anion
4K
3K
5K
ðPO3K
4 ; P2 O7 ; P3 O9 ; and P3 O10 Þ release. All ion
release studies were carried out using ion chromatography (Dionex, Surey, UK) except for Sr2C where
inductively coupled plasma mass spectrometry (ICPMS, Spectromass 2000, Kleve, Germany) was used.
Owing to the much greater expected concentration
of ions, samples were diluted in deionized water
(18 MU cmK1). The final cumulative concentration was
calculated from the results of the measurements taking
into account the dilution factor (Ahmed et al. 2005).
2.3.5. pH measurements. At each time point, pH
measurements were taken after transferring the glass
discs to a fresh solution (deionized water, pH 7G0.2).
The measurements were collected using a Hanna
Instruments pH 211 Microprocessor pH meter (BDH,
UK) with attached glass combination pH electrode
(BDH, UK). The pH electrode was calibrated using
pH calibration standards (Colourkey Buffer Solutions.
BDH, UK). The degradation studies and preparation
of the standard solutions described above were conducted using high-purity water. This was obtained
from a PURELAB UHQ-PS system (Elga Labwater,
UK), which purified the water to a level of
18.2 MU cmK1 resistivity.
2.4. Substrate surface characterization
2.4.1. Wettability and SFE. Assessment of the surface
wettability of the glasses was performed using the
sessile drop method using a KSV Optic Contact Angle
Meter CAM200. Contact angle measurements were
conducted at room temperature with three different
liquids: deionized water; diiodomethane (Sigma); and
formamide (Sigma). Prior to the measurements, the
surface of the samples was polished to a mirror finish
using SiC paper (No. 4000) to avoid the influence of the
roughness on the wettability and then cleaned with
methanol in an ultrasonic bath for 10 min before drying
with compressed air. Measurements for each liquid/
sample combination were performed in triplicate.
SFEs were calculated from the contact angle using
two methods: geometric mean method (Owens–Wendt,
OW) and the acid–base method (van Oss–Good, VO).
For all samples, dispersive (gd, gLW), polar (gp), acid–
base (gab), acidic (ga) and basic (gb) components
were calculated.
2.4.2. Cell viability. Human osteosarcoma cells (HOS)
were used to obtain a preliminary estimate of biological
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438 Strontium-doped phosphate-based glasses
E. A. Abou Neel et al.
(a)
(b)
0
–10
–20
–30
– 40
(c)
200
100
0
–100
–200
(d )
7.05 T
simulation
14.1 T
simulation
50
0
–50
–100
–150
–200
ppm
0
–50
–100
–150
–200
ppm
Figure 1. (a) The overlaid 31P MAS NMR spectra obtained from the glass samples, with the horizontal frequency scale expanded
to show only the isotropic region, (b) fit of the 31P MAS NMR spectrum obtained from the CNP glass, with spinning sidebands
visible on either side of the centre bands, (c) the 23Na MAS NMR spectra obtained from the PBGs at 7.05 T and (d ) experimental
and simulated spectra of the CNP glass at 14.1 and 7.05 T. (a,c,d ) Solid line, CNP; dotted line, CNPSr1; dashed line, CNPSr3;
dot-dashed line, CNPSr5.
compatibility of these glasses using live–dead staining
and confocal laser scanning microscopy (CLSM, BioRad, USA) as described previously (Abou Neel et al.
2007; Abou Neel & Knowles 2008).
2.5. Statistical analysis
The Student’s t-test (Abou Neel et al. 2007) was used to
analyse the significance of changes in density and the
glass transition temperature as a function of SrO
content. Significance was set at a 0.05 level, and all
statistical analyses carried out using the SPSS system
for WINDOWS (SPSS v. 12.0.1).
3. RESULTS
3.1. Structural characterization
3.1.1. X-ray powder diffraction. A sodium calcium
phosphate (NaCa(PO3)3-23-669) phase was detected
with XRD for all glass compositions, after crystallization. Strontium phosphate (Sr3(PO4)2-32-1251) was
also identified as a minor phase in the CNPSr5 glass.
3.1.2. Nuclear magnetic resonance. Figure 1a shows the
overlaid 31P MAS NMR spectra obtained from the glass
samples, with the horizontal frequency scale expanded
J. R. Soc. Interface (2009)
about the isotropic region such that the spinning
sidebands are not shown. Each spectrum was fitted
using Gaussian peaks centred at approximately K8,
K24 and K32 ppm, representing Q1, Q 2 and Q 3
phosphorus environments, respectively (Qn denoting a
phosphate tetrahedron with n bridging oxygens). In
order to obtain the relative abundances of each of these
Qn species, the intensities of these peaks were fitted
(along with the spinning sidebands). An example of
such fitting is shown in figure 1b and the results of such
fitting are given in table 2.
Figure 1c shows the overlaid 23Na MAS NMR
spectra obtained from the glasses. All the spectra
show a single broad peak that remains effectively
constant as the SrO content increases. This shape is
characteristic of sodium observed in a glass where there
is a spread of quadrupolar interactions (Kohn et al.
1998; Smith & Van Eck 1999). The multiple field data
can be used to constrain things since under MAS there
are effectively two residual interactions affecting the
observed lineshape. There are residual second-order
quadrupole effects and the spread of isotropic chemical
shifts, often termed the chemical shift distribution.
These interactions are, respectively, inversely and
directly proportional (Hz) to the applied magnetic
field (Smith & Van Eck 1999). The simulation employs
a Gaussian distribution in the quadrupolar coupling
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Strontium-doped phosphate-based glasses
31
P MAS NMR peak parameters.
140
120
glass
code
Qn
species
shift
abundance
(G0.5 ppm) (G1%)
CNP
1
2
3
1
2
3
1
2
3
1
2
3
K8.4
K24.1
K32.9
K8.2
K24.1
K32.1
K8.4
K24.1
K31.4
K8.3
K24.4
K31.6
CNPSr1
CNPSr3
CNPSr5
1.2
94.5
4.4
1.1
93.9
5.0
2.3
92.1
5.6
2.3
91.6
6.2
linewidth
(G1 ppm)
6.7
10.0
11.1
7.0
9.8
10.6
8.6
9.8
9.4
8.5
10.0
10.2
transmission (%)
Table 2. The
E. A. Abou Neel et al. 439
100
(d)
80
(c)
60
(b)
40
(a)
20
0
1400
1200
1000
wavenumber
800
600
(cm–1)
Figure 2. FTIR spectra: (a) CNP, (b) CNPSr1, (c) CNPSr3
and (d) CNPSr5.
constant CQ (MacKenzie & Smith 2002; full-width halfmaximum (FWHM) of this distributionZDCQ), which
can be used as a semi-quantitative measure of disorder
(O’Dell et al. 2007). An example of such fitting is shown
in figure 1d. The spectra were simulated with a mean
CQZ2.4G0.1 MHz, DCQZ2.9G0.1 MHz, and an isotropic chemical shift of K8G0.5 ppm. These parameters gave a good fit to all of the 23Na MAS NMR
lines in figure 1c. For simplicity, the isotropic chemical
shift was kept constant, and the asymmetry parameter
hQ was set at 0, although in reality a distribution in CQ
would probably mean a distribution in these parameters also.
3.1.3. FTIR spectroscopy. Figure 2 shows the FTIR
spectra from the CNPSr1, CNPSr3 and CNPSr5
samples compared with CNP. As can be seen, the
spectra from the strontium-containing glasses show
only very subtle variation as a function of composition.
However, the main change observed is a reduction in
the intensity and a slight shift to higher wavenumber of
the nas(PdOdP) mode (at approx. 900 cmK1) as the
Sr2C content increases. The other noticeable change is
a general broadening of the spectral features as Na2O is
replaced by SrO. Generally, the absorption bands in the
above spectra can be assigned according to the data in
table 3 (Byun et al. 1995; Moustafa & El-Egili 1998;
Ilieva et al. 2001; Shih & Shiu 2007).
3.1.4. Elemental analysis. The O, P, Ca, Na and Sr were
detected from all the strontium-containing glass
samples. These elements were also calculated as oxides
and considered in mol% as given in table 4. The analysis
showed that there is a specific trend for the amount of
Sr incorporated in the structure with less than 1 per
cent error. This trend correlated with the actual Sr
content included in the glass structure. It was also
observed that the weight% of both P and Ca remained
relatively constant for all studied glass composition.
The weight% of Na, however, is changing with the
composition. From the calculated oxides, it seems
that both P2O5 and SrO are as expected; however,
there is more Na2O and less CaO than expected
particularly for CNPSr1 and CNPSr3. Table 5
J. R. Soc. Interface (2009)
Table 3. Infrared band assignments for the Sr-containing
PBGs (d, deformation; n, stretching; s, symmetric; as,
asymmetric).
assignment; Byun et al. (1995),
associated
Moustafa & El-Egili (1998),
wavenumber Ilieva et al. (2001) and O’Dell Qn (where
et al. (2007)
(cmK1)
applicable)
540
725
770
900
1000
1100
1270
d (PdOdP)
ns(PdOdP)
ns(PdOdP)
nas(PdOdP)
ns(PO3)2K
nas(PO3)2–
nas(PO2)–
Q2
Q1
Q1
Q2
compares the average number of non-bridging oxygen
atoms per PO3K
4 (NBO/P) calculated from the compositions with those calculated from the NMR
Q-speciation. These ratios were calculated using
the equations
NBO
ð2cCa C cNa C 2cSr Þ
Z
;
ð3:1Þ
P
cP
composition
where ci represents the concentration of element i, and
NBO
Z ½Q 2 C 2½Q 1 ;
ð3:2Þ
P
NMR
where Qn is the fraction on Qn species. Note that these
NBO/P ratios do not include the terminal P]O group.
Table 4. Glass composition as obtained from energy dispersive
X-ray spectroscopy.
composition (mol%)
glass
code
Na2O
P2O5
CaO
SrO
CNP
CNPSr1
CNPSr3
CNPSr5
24.3G2.2
26.2G0.6
28.3G1.6
18.9G1.1
51.0G3.8
47.7G3.5
45.8G4.3
46.0G4.9
24.8G1.6
24.0G1.9
20.8G1.1
23.7G2.9
—
2.0G1.1
5.2G1.6
11.5G1.0
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E. A. Abou Neel et al.
NBO/P calculated from NBO/P calculated
composition G15%
from NMR G1%
CNP
CNPSr1
CNPSr3
CNPSr5
0.96
1.10
1.18
1.18
0.97
0.96
0.97
0.96
3.2. Bulk glass characterization
3.2.1. Density. Table 1 gives the effect of replacement of
Na2O with SrO on the density of the glasses. The results
show that introducing SrO into the glass causes an
increase in density that correlates well with the SrO
concentration. It should be noted, however, that the
difference in density between the CNP and CNPSr1
samples is not statistically significant.
3.2.2. Thermal characterization. Table 1 gives the glass
transition temperatures of the PBGs obtained from
the DSC curves. As observed for the density, the Tg
of the glasses also increased with increasing SrO
content. Again, it should be noted that the differences
between the Tg values for the CNP and CNPSr1
samples, and the CNPSr3 and CNPSr5 samples, are not
statistically significant.
DTA provides an overview of the possible transformations that the glasses undergo at different temperatures, such as the glass transition (manifested by the
first inflection in the trace), crystallization temperature
(characterized by exothermic peak) and melting
temperature (characterized by endothermic peak).
Figure 3a,b shows the DTA traces from all three glass
compositions. For CNP glass (figure 3a), the trace was
characterized by the presence of a single sharp crystalline peak at 6008C and two melting peaks at 757 and
8648C. Crystallization of the CNPSr1 glass occurred at
6508C and the two melting peaks were identified at 756
and 8668C. For CNPSr3 glasses, the crystallization
peak shifted to a higher temperature (6838C) and the
melting peaks were observed at 750 and 8948C.
Crystallization was further retarded for the CNPSr5
glasses (7208C). Furthermore, a second crystallization
event was observed at 7508C. The melting peak for this
composition was also identified at higher temperature
(9108C; figure 3b).
3.2.3. Degradation. Figure 4 shows the cumulative
degradation as weight loss per unit area as a function
of time for all glass compositions. The weight loss per
unit area increased with time for all samples, and the
rate of weight loss increased significantly with increasing SrO. For the purpose of comparison, the
degradation rates were estimated by fitting the
degradation curves with a straight line. Using this
method, values of 6.4G0.25, 29.55G4.03, 19.0G4.34
and 14G4.46 ! 10K3 % mmK2 hK1 were obtained for
J. R. Soc. Interface (2009)
Tc
endothermic down
glass code
(a)
Tg
CNP
Tm1 Tm2
(b)
endothermic down
Table 5. Average number of non-bridging oxygen atoms per
PO3K
4 calculated from the elemental analysis compared with
those calculated from the 31P NMR results. (Note that these
NBO/P ratios do not include the terminal P]O group.)
Tc
Tg
Tg
Tc
Tg
CNPSr5
Tc
CNPSr3
CNPSr1
Tm
200
300
400
500 600 700
temperature (°C)
800
900
1000
Figure 3. (a,b) DTA traces from PBGs doped with different
concentrations of strontium oxide. (a) Solid line, 0 mol% TiO2.
0.6
cumulative degradation
(% mass loss/SA)
440 Strontium-doped phosphate-based glasses
0.5
0.4
0.3
0.2
0.1
0
2
4
6
8
10
time (days)
12
14
16
Figure 4. Degradation profiles (i.e. presented as cumulative
weight loss %/surface area) for the CNPSr1 (opened circles),
CNPSr3 (filled down triangles) and CNPSr5 (open up
triangles) glasses compared with the CNP (filled circles)
control glass as a function of time.
the CNP, CNPSr1, CNPSr3 and CNPSr5 compositions, respectively. Interestingly, the degradation rate
increased with the addition of 1 mol% SrO and then
decreased with further addition of SrO.
3.2.4. Cumulative ion release measurements.
Cumulative cation release measurements The release
profiles for NaC ions follow exactly the same trend as
the degradation with the CNPSr1 composition showing
the highest level of release followed by the CNPSr3,
CNPSr5 and CNP compositions in that order. For the
Ca2C release, however, the CNPSr1 composition
showed the highest level of release followed by the
CNP, CNPSr3 and CNPSr5 compositions in that order.
It was also observed that the CNPSr1 composition
released the highest levels of both NaC and Ca2C
Downloaded from http://rsif.royalsocietypublishing.org/ on June 15, 2017
Strontium-doped phosphate-based glasses
cations. The release rates were estimated by fitting a
straight line of the form yZmx through the data.
The estimated release rates for Sr2C also correlated
well with the SrO content of the glasses (figure 5).
Cumulative anion release measurements The
cumulative release of four different anionic species:
orthophosphate ðPO3K
4 Þ; cyclic trimetaphosphate
pyrophosphate ðP2 O4K
and linear
ðP3 O3K
7 Þ;
9 Þ;
polyphosphate ðP3 O5K
Þ
was
identified.
Of these
10
was
released
in
the
largest
quantities
anions, P3 O3K
9
5K
4K
followed by PO3K
4 , P3 O10 and P2 O7 in that order. The
release rate for each anionic species was also calculated
by linear fitting of the data and presented in figure 5.
The Sr-containing glasses all exhibited higher levels of
4K
PO3K
4 and P2 O7 release than the CNP control glasses.
anion, the Sr-containing
In the case of the P3 O3K
9
glasses all exhibited a lower release rate than the CNP
control glasses. The CNP and CNPSr5 compositions
released P3 O5K
10 ions at the highest and lowest rates,
respectively, with the CNPSr1 and CNPSr3
compositions releasing this anion at approximately
the same rate midway between those of the other
two compositions.
3.2.5. pH analysis. Figure 6 shows the pH changes of the
deionized water used for the degradation studies as a
function of time for all glass compositions. For all
samples, the dissolution medium reached a constant pH
after 4 days. CNPSr1 glasses displayed the minimum
(4.2) pH value of the surrounding medium. The lowest
reduction in pH was observed for CNP glasses where
the medium reaches a pH of 4.92G0.06. The pH for the
medium surrounding the CNPSr3 and CNPSr5 glasses
settled at approximately 4.6.
3.3. Substrate surface characterization
3.3.1. Wettability and SFE. Results of SFE calculated
on the basis of the OW method (KSV Instruments
2007) are shown in figure 7a. The total SFE for all
samples was 70 mN mK1. The dispersive component
(gd) was approximately 37 mN mK1 for CNP and
CNPSr5 samples, while for CNPSr1 and CNPSr3
glasses, it was 34 mN mK1. The polar component (gp)
was at the level of 34 mN mK1 for CNP and CNPSr5
samples and 36 mN mK1 for CNPSr1 and CNPSr3
samples. In addition, the fractional polarity (FP) was
calculated as FPZgp/(gp/gd; Ponsonnet et al. 2003;
figure 7b), but no significant variations were observed
between samples.
SFE obtained from the VO method (KSV Instruments 2007) showed that the dispersive component
(gLW) was the highest for CNP and CNPSr5 glasses
(approx. 44 mN mK1) as shown in figure 7c. These
glasses also had the lowest acidic (ga 0.55 and
0.65 mN mK1, respectively) and acid–base components
(gab 8.5 and 10 mN mK1, respectively) as shown in
figure 7d. For the CNPSr1 and CNPSr3 glasses, these
components were as follows: acidic (ga) 0.79 and
0.87 mN mK1, respectively, and acid–base (gab) 12.1
and 13.2 mN mK1, respectively, as shown in figure 7d.
J. R. Soc. Interface (2009)
E. A. Abou Neel et al. 441
3.3.2. Cell viability. Figure 8 shows CLSM images
of live/dead stained HOS attached to the surfaces of
the tested glass compositions and the positive control
after 24 hours of culture. As can be seen from these
images, the cells showed relatively better response on
strontium-containing glass surfaces than strontiumfree glasses. This could be detected from both the
number and morphology of cells where higher number
of cells were observed on the surface of strontiumcontaining glass, particularly CNPSr1 and CNPSr3.
The cells had rounded morphology when they attached
to CNP glasses compared with the more flattened
morphology when they grow on all strontiumcontaining glasses. Moreover, both CNP and CNPSr5
showed lower number of cells attached to both surfaces.
However, CNPSr1 and CNPSr3 showed relatively
more cells attached to their surfaces in a manner
comparable to the positive control up to the time of
the experiment. After 24 hours, no cells were detected
on all glass surfaces.
4. DISCUSSION
Sr2C is known to behave in a comparable way to
Ca2C, which has similar ionic size and polarity to
Ca2C ( Wren et al. 2008). Its role in bone metabolism
has been well documented; it has both anti-resorptive
and bone-forming effects that can be used to decrease
the risk of fracture in postmenopausal women when
administered as SR, marketed as Protelos. In the form
of SR, Sr2C can induce bone cell replication and
collagen synthesis, while also inhibiting the activity of
osteoclasts (Marie 2005).
The purpose of this study was to investigate the
possibility of incorporation of Sr2C into PBGs for
potential applications in bone tissue engineering. The
effect of incorporation of Sr2C on the glass structure,
degradation, associated ion release and pH changes that
affect the surrounding environment were studied.
The XRD data revealed that, after crystallization by
heating to Tc, all the glass compositions contained the
same phase (NaCa(PO3)3-23-669); the only strontiumcontaining phase (Sr3(PO4)2-32-1251) was identified as
a minor component in the CNPSr5 sample.
The dominance of the Q 2 peak in the 31P MAS NMR
spectra was to be expected since phosphate glasses
containing 50 mol% P2O5 are well known to consist
almost entirely of infinite chains or rings of phosphate
tetrahedra (Brow et al. 1990). Increasing the SrO
content resulted in a decrease in Q 2 species in the
glasses with both the Q1 and Q 3 species increasing. This
is perhaps evidence of disproportionation of Q 2 species
according to the relationship 2Q 2/Q1CQ 3. Such
disproportionation has previously been observed in
metaphosphate glasses containing sodium and calcium
(Fletcher et al. 1993; Velli et al. 2005), and, in this case,
shows a slight increase with SrO content. No linear
trend in either the chemical shift or the linewidth of the
31
P MAS NMR peaks occurs as the Sr2C content was
increased. However, the lack of any change in the 23Na
MAS NMR spectra indicates that the Sr2C had no
appreciable effect on the average sodium environment.
Downloaded from http://rsif.royalsocietypublishing.org/ on June 15, 2017
0.030
0.025
0.020
0.015
0.010
0.005
0
E. A. Abou Neel et al.
140
35
120
30
100
25
20
80
15
60
10
40
5
20
0
0
CNP
Na+
P3O93 –
Ca2+, PO43– P2O74 –, Sr2+
release rate (ppm h–1)
0.035
Na+, P3O93– release (ppm h–1)
degradation rate (% mm–2 h–1)
442 Strontium-doped phosphate-based glasses
Ca2+
PO43 –
P2O74 –
P3O105 –
Sr2+
degradation
CNPSr1 CNPSr3 CNPSr5
glass code
Figure 5. Rate of degradation, and ion release for different cationic and anionic species as a function of the glass composition. The
rate was estimated by fitting a straight line of the form yZmx through the data.
J. R. Soc. Interface (2009)
8
6
pH changes
FTIR spectra from the strontium-containing glasses
showed only very subtle variation as a function of
composition: this was to be expected since substitution
of Na2O by SrO does not change the O/P ratio and
hence no variation in the Q-speciation was expected,
and the variation determined from the 31P NMR is only
slight. However, the cause of the observed shift in the
frequency of the nas(PdOdP) mode is the replacement
of NaC ions by higher field strength Sr2C ions (Shih &
Shiu 2007). A previous neutron diffraction study of
CaO–Na2O–P2O5 glasses (Pickup et al. 2007) demonstrated that increasing the average cation field strength
by replacing NaC with Ca2C caused a shortening of the
phosphorus to bridging oxygen distance. The FTIR
results suggest that a similar effect is happening here
with the replacement of NaC by Sr2C causing a
shortening of the PdOdP bonding, which results in
a shift in the frequency of the nas(PdOdP) mode to
higher energy. The general broadening of the spectral
features with Sr2C content is most probably due to the
increased variation of the phosphate–cation bonding
interactions caused by the introduction of the third
cation. In fact, comparing the FTIR spectrum from the
CNP glass with those from the Sr-containing glasses, it
can be seen that the absorption bands in the latter
spectra are broader and less well resolved. This suggests
that addition of just 1 mol% SrO causes a significant
increase in disorder in bonding; it is perhaps this
increased disorder that causes the increased solubility
of the Sr-containing glasses compared with the CNP
glass. Glasses are metastable with respect to the
crystalline state; they are also disordered relative to
their crystals. From this, it can be inferred that there is
some instability associated with disorder, since the
solubility is a trade-off between the energy required to
dissociate the structure and the energy released upon
hydration of the resultant ions. Therefore, it seems
reasonable to suggest that a more disordered, less stable
structure may be more soluble.
The NBO/P ratios calculated from the elemental
analysis are in close agreement with those calculated
from the 31P NMR results, if one takes into consideration the errors associated with the two sets of data.
The Sr-containing glasses, CNPSr1 and CNPSr3 in
particular, showed higher Na2O than expected, and this
4
2
0
2
4
6
8
10
time (days)
12
14
16
Figure 6. Changes in pH of deionized water as a function of
time for the CNPSr1 (open circles), CNPSr3 (filled down
triangles), and CNPSr5 (open up triangles) glasses compared
with the CNP (filled circles) control glass.
could also explain the fact that these glasses showed a
higher degradation rate than Sr-free glasses.
The observed increase in density with incorporation
of SrO into the glass structure could be attributed to
the much higher density of SrO (4.7 g cmK3) than that
of Na2O (2.27 g cmK3). This increase in density also
correlated with an increase in the Tg and coupled with a
shift in both crystallization and melting peaks to higher
temperature. However, incorporation of SrO increased
the degradation rate of these glasses. This was not
expected since replacing Na2O with CaO in the CaO–
Na2O–P2O5 system effectively reduces the degradation
(Franks et al. 2000). Incorporation of only 1 mol% of
SrO produced a fivefold increase in the degradation
compared with glasses free from SrO. With further addition of SrO, the solubility is reduced but still remains
significantly higher than that of SrO-free glasses.
The results of the ion release study here reflected the
degradation behaviour of the glasses with all the SrOcontaining glasses exhibiting greater ion release than
the SrO-free glasses as shown in figure 5. Comparing the
release of different cations, NaC ions were released at
higher levels than Ca2C. It is notable that even though
the CNP glass contained the highest Na2O content,
it released less NaC than the SrO-containing glasses.
Downloaded from http://rsif.royalsocietypublishing.org/ on June 15, 2017
Strontium-doped phosphate-based glasses
(b) 0.6
0.5
60
0.4
FP
SFE (mN m–1)
(a) 80
E. A. Abou Neel et al. 443
40
0.3
0.2
20
0.1
(c)
0
0
60
(d ) 18
16
14
12
10
8
6
4
2
0
50
40
30
20
10
0
Sr0
Sr1
Sr3
sample code
Sr5
Sr0
Sr1
Sr3
sample code
Sr5
Figure 7. Results of SFE calculations using the OW method: (a) SFE and its dispersive and polar components (black bars, SFEd;
light grey bars, SFEp; dark grey bars, SFEtot) and (b) FP. Results of SFE calculation using VO method: (c) total and dispersive
component of the SFE (black bars, gd; light grey bars, g) and (d ) acidic, basic and acid–base components of the SFE (black bars,
V 000 ga (V 000 mN mK1); light grey bars, V 000 gb (V 000 mN mK1); dark grey bars, gab (mN mK1).
(a)
(b)
100 µm
(c)
100 µm
(d)
(e)
100 µm
Figure 8. Confocal images of both controls ((a) CNP and (e) Thermanox) compared with those showing viability of HOS cells
seeded on the surface of glass discs of (b) CNPSr1, (c) CNPSr3 and (d ) CNPSr5 after 24 hours of culture.
These results suggest that the dominant factor affecting
NaC release is the overall degradation rate of the glass,
not the Na2O content. Hence, since the CNPSr1 glass
showed the highest degradation rate, it also exhibited
the highest level of NaC release. The Ca2C ion release
followed a similar trend to the NaC ion release.
Addition of 1 mol% SrO increased the Ca2C release
relative to the CNP glass, but further additions of SrO
of 3 and 5 mol% reduced the rate of Ca2C release to
below that of the SrO-free glass. These results suggest
that, as with the NaC release, the release of Ca2C ions is
strongly influenced by the overall degradation rate of
the glass. By contrast, the release of Sr2C ions was
strongly correlated with the SrO content of the glasses.
Regarding the anion release data, the highest release
was observed for the P3 O3K
anion. This suggested
9
that a significant concentration of the P3 O3K
9 anion was
present in the original sample. The lowest release was
observed for the P2 O4K
7 ions; similar results have been
reported in previous work (Abou Neel et al. 2005a;
Abou Neel & Knowles 2007). In general, the release of
all the anionic species measured followed the same
trend as the glass degradation.
J. R. Soc. Interface (2009)
The changes in degradation associated with the glass
composition were reflected not only in the concentration and rate of ions released but also in the changes
in pH of the surrounding medium. As mentioned
previously, the CNPSr1 glasses, having the highest
degradation rate, displayed the greatest decrease
(4.92G0.06) in pH of the surrounding medium from
the initial value of 7G0.1. Moreover, the smallest
reduction in pH was observed for the CNP glasses that
showed the lowest degradation rate while both CNPSr3
and CNPSr5 glasses caused an intermediate pH change
compared with the other compositions.
Numerous studies have demonstrated that the
wettability of the surface has an influence on biological
properties of biomaterials and cellular behaviour on the
surface (Ponsonnet et al. 2003; Wirth et al. 2005, 2008;
Yeung et al. 2008). However, it was shown to be more
important to compare SFE and its components
(especially the polar one), since they correlate to the
cell adhesion and proliferation. Maximum cellular
adhesion was previously observed when FP was equal
to 0.3 (Hallab et al. 1995), and it was also shown that
the lower the FP, the better the fibroblast proliferation.
Downloaded from http://rsif.royalsocietypublishing.org/ on June 15, 2017
444 Strontium-doped phosphate-based glasses
E. A. Abou Neel et al.
Furthermore, poor cell spreading was observed on low
SFE materials (Schakenraad et al. 1988). In this study,
the highest total SFE and the lowest FP were observed
for CNP and CNPSr5 glasses. However, FP values were
much higher than 0.3, and also they still showed no
significant differences from CNPSr1 and CNPSr3
regarding the SFE and FP. This suggests that these
glasses may not have surfaces that will encourage cells
to attach and proliferate. This finding was also
confirmed by the cell viability study, where these
glasses showed lower or comparable viability up to
24 hours of culture, but, after that period, no cells could
be detected. Regardless of these findings, the incorporation of strontium into these glasses improved the
cellular response by increasing the number of cells
attached to the surface and this also can be confirmed
from the morphology of cells. Therefore, the reduced
viability could not be due to the presence of strontium
but was due to the very high degradation nature of
these glasses, since the glass surfaces are not stable for
cells to attach. Results obtained from both OW and VO
methods were in agreement. Higher dispersive and
lower polar components were observed for the CNP and
CNPSr5 glasses. However, the basic component of the
results of the acid–base calculation was the same for all
samples, although differences were observed in the acid
component, which was lower for the CNP and CNPSr5
samples. The acid components for all of the samples
were significantly lower than that of the base, which
suggested low acidic character of the surface. It can be
concluded that from a thermodynamic point of view
addition of SrO had no significant effect on the
SFE components.
5. CONCLUSIONS
Substitution of Na2O with SrO from 0 to 5 mol%
produced a significant increase in density, glass
transition temperature and degradation rate of these
glasses. The increase in degradation rate was also
confirmed by the levels of both cation (NaC and Ca2C)
4K
3K
5K
and anion ðPO3K
4 ; P2 O7 ; P3 O9 ; and P3 O10 Þ release
from these glasses, and in turn by changes in the pH of
the surrounding medium. Sr2C release, however, was
directly related to the amount of SrO in the glass and
not to the degradation rate.
31
P MAS NMR results showed that, as the Sr2C
content was increased, the amount of Q 2 phosphorus
decreased slightly, resulting in a slight increase in the
number of Q1 and Q3 environments. 23Na MAS NMR
results suggested that the sodium environment was not
significantly affected by increasing the Sr2C content. A
shift in the FTIR frequency of the nas(PdOdP) mode
to higher energy has been observed with the replacement of NaC with higher field strength Sr2C ions, which
is associated with the shortening of the phosphorus to
bridging oxygen distance and consequent strengthening
of this bond. Moreover, the general broadening of the
spectral features with Sr2C content is most probably
due to the increased variation of the phosphate–cation
bonding interactions caused by the introduction of the
third cation. It is perhaps this increased disorder or the
fact that the Sr-containing glasses have higher Na2O
J. R. Soc. Interface (2009)
than expected that causes the increased solubility of
these glasses compared with the CNP glass.
Analysis of thermodynamic aspects of the surfaces,
based on SFE evaluations, revealed no significant
influence of strontium on SFE components, especially
FP. However, incorporation of Sr2C produced glasses
with better cellular response than Sr2C-free glasses,
but relatively lower cellular response than the positive
control. This lower response could be due to the high
degradation nature of these glasses and not due to the
presence of strontium since the Sr2C-free glasses
showed lower degradation but still showed the worst
cellular response. Therefore, in our future work, we will
tailor the chemistry to get glasses with relatively lower
degradation and still incorporating Sr2C as a means of
improving the biocompatibility.
The authors would like to acknowledge the EPSRC (Grant
nos. EP/C004671/1 & EP/C004698/1 & EP/C515560/1) for
providing the funds to conduct this work. M.E.S. thanks the
EPSRC and University of Warwick for partially supporting
the NMR equipment at Warwick.
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